1. Field of the Invention
The present invention relates to chemiluminescent compounds. More particularly, the present invention concerns stable, triggerable chemiluminescent 1,2-dioxetanes. Even more particularly, the present invention concerns new chemiluminescent 1,2-dioxetane compounds derived from the oxidation of novel alkenes prepared by the coupling of substituted aromatic esters or ketones and spiro-fused ketones with or without a T-electron system or a carbon-carbon double bond in the ring.
2. Prior Art
Chemiluminescent compounds, their preparation and their uses have been long documented in the prior art. These xe2x80x9chigh energyxe2x80x9d molecules store sufficient energy to generate, or fragmentation, electronically excited carbonyl products which are responsible for the observed chemiluminescence. Dioxetanes and especially, 1,2-dioxitanes and eminently useful to detect the presence, as well as the absence, of certain enzymes in fluids such as blood and the like because of their chemiluminescence. Thus, 1,2 dioxetanes are eminently useful in doing medical assays.
Generally, 1,2-dioxetanes are thermally labile substances having a wide range of stability which decompose on heating and emit light, and correspond to the following formula (1): 
Where each R corresponds to any one of a multitude of organic moieties widely reported in the prior art, as detailed herebelow. As noted these 1,2-dioxetanes have a wide range of stability. For example, the prior art, as found in: (a) K. W. Lee, L. A. Singer and K. D. Legg, J.Org.Chem., 41, 2685(1976); (b) F. McCapra, I. Beheshti, A. Burford, R. A. Hanu and K. A. Zaklika, J.Chem.Soc.,Chem.Commun., 944(1977); and (c) J. H. Wieringa, J. Strating, H. Wynberg and W. Adam, Tet. Lett., 169 (1972); respectively, disclose the following 1,2-dioxetanes of different stability: 
Although these high energy compounds are all spiro-substituted 1,2-dioxetanes, spiroadamantane substitution exerts a tremendous stabilizing effect on these four-membered ring peroxides. The lower activation energy (EA) of the dioxetanes of formulae (3) and (4) above is explained by the donation of charge from nitrogen to the dioxetane ring. The dioxetane of formula (5) above decomposes at 150xc2x0 C. and has a half life at 25xc2x0 C. more than 20 years.
A 1,2-dioxetane (6) below, dispiro[adamantane2,3xe2x80x2-[1,2-]dioxetane-4xe2x80x2,9xe2x80x2-fluorene] was isolated as crystals and described by W. Adam and L. A. A. Encarnacion, Chem. Ber., 115, 2592 (1982). 
The stability of 1,2-dioxetanes (5) and (6) was described on the basis of bulky and rigid spiro nature of the adamantane group.
The first stable and enzymatic triggerable 1,2-dioxetane was synthesised by the oxidation of (6-acetoxy-2-naphthyl) methoxy methyleneadamantane as reported by A. P. Schaap, R. S. Handley and B. P. Giri, Tet. lett., 935 (1987). This 1,2-dioxetane utlizes aryl esterase emzyme to catalyze the cleavage of the acetate group of a naphthylacetate-substituted-1,2-dioxetane and produce chemiluminescence in aqueous buffers at ambient temperature by the following sequence: 
Several other stabilized 1,2-dioxetanes and their use as enzyme substrates have been disclosed in the literature. See, inter alia, A. P. Schaap, T. S. Chen, R. S. Handley, R. DeSilva and B. P. Giri, Tet. Lett., 1155(1987); A. P. Schaap, M. D. Sandison and R. S. Handley, Tet. Lett., 1159 (1987); U.S. Pat. Nos. 4,962,192; 4,978,614; 5,386,017; 5,721,370, the disclosures of which are hereby incorporated by reference.
These several other 1,2-dioxetanes, generally, have the following general structures: 
wherein 
is a non-active site and which is selected from the group of polycyclic alkyl groups containing 6 to 30 carbon atoms, OX is an oxy group substituted on an aryl ring which forms an unstable oxide intermediate 1,2-dioxetane compound when triggered to remove X by an activating agent selected from the group consisting of an acid, a base, a salt, an enzyme and an inorganic or organic catalyst, and electron donor source, and X is a chemically labile group which is removed by the activating agents to form light and carbonyl containing compounds, R1 is a lower alkyl containing 1 to 8 carbon atoms, or mixtures thereof, or 
where T is a non-active site which is a cycloalkyl or a polycycloalkyl group bonded to the 4-membered ring portion of the dioxetane by a spiro linkage; Y is a fluorescent chromophore; X is a hydrogen, alkyl, aryl arylkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, or enzyme cleavable group; and Z is hydrogen or an enzyme cleavable group, provided that at least one of X or Z must be an enzyme cleavable group.
The enzyme cleavable 1,2-dioxetanes of formulae (14), (15) and (16) shown below have been commercialized and used in immuno assays, southern blotting, northern blotting, western blotting, plaque/colony lifts and DNA sequencing. 
The 1,2-dioxetane of formula (15) with the chloro substitution in the adamantane ring demonstrates better results in DNA sequencing when compared to dioxetane (14). Dioxetane (16) is more soluble in an aqueous system than 1,2-dioxetane (14) and (15) when a CH3 group is replaced with a CH2CH2CH2COOH.
Other relevant prior art can be found in U.S. Pat. Nos. 5,386,017; 4,962,192; 5,018,827; 5,578,253; 5,004,565; 5,068,339, the disclosures of which are hereby incorporated by reference.
While these prior art compounds provide enzyme cleavable 1,2-dioxetanes, it has been observed that in an aqueous buffer, the luminescence of these molecules is particularity poor, especially when trace amounts of biological materials are sought to be detected. Thus more powerful dioxetanes are needed i.e. dioxetanes having higher levels of chemiluminescence in an aqueous buffer.
The present invention provides novel 1,2-dioxetanes derived from spiro-fused ketones with or without xcfx80-electrons in the ring or with carbon-carbon double bond(s) in the spiro-fused ring. Additionally, these new dioxetanes have electron donating or withdrawing groups at the four-membered peroxide ring to render these dioxetanes active at all sites.
The 1,2-dioxetanes hereof generally correspond to the formula: 
wherein
(1) when Arxe2x80x94Oxe2x80x94Y and OR join together to give an aryl group substituted with an X-oxy group to form a stable 1,2-dioxetane intermediate which is triggerable to form an unstable intermediate oxide, R2 and R3 either form 
xe2x80x83which is either a cyclic, polycyclic or spiro-fused ring containing at least one carbon-carbon double bond or carbon-carbon triple bond in the ring or side chain with or without heteroatoms or 
xe2x80x83which is a cyclic, polycyclic or spiro-fused ring containing substituted or unsubstituted fused aromatic ring or substituted or unsubstituted aromatic ring attached by linker arms; or
(2) when Arxe2x80x94Oxe2x80x94Y and OR, do not join together
(a) Ar is aryl and may be phenyl, substituted phenyl, naphthyl, substituted naphthyl, anthryl, substituted anthryl or other aromatic or nonaromatic fluorescent or nonfluorescent group; Y is a hydrogen, alkyl, acetate, t-butyldimethylsilyl or an enzyme cleaveable group, an antibody cleaveable group; R1 is selected from the group consisting of alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)2, alkyl(etheralkyl)3, alkyletherhaloalkyl, alkyl(etherhaloalkyl)2, alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenated alkyl(mono, di, tri or any position in normal or branched or cyclic chain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono or dibasic) or the inorganic salts thereof, haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) or inorganic salts, linker-flourescent molecule, linker-antibodies, linker-antigen, linker-biotin, linker-avidin, linker-protein or linker-carbohydrates or linker-lipids; when R2 and R3 forms either 
xe2x80x83which is a cyclic, polycyclic or spiro-fused ring containing at least one carbon-carbon double bond or cabon-carbon triple bond in the ring or side chain with or without heteroatoms, or 
xe2x80x83which is a cyclic, polycyclic or spiro-fused ring containing substituted or unsubstituted fused aromatic ring or substituted or unsubstituted aromatic rings attached by linker arms, or
(b). Ar is aryl and may be phenyl, substituted phenyl, naphthyl, substituted naphthyl, anthryl, substituted anthryl or other aromatic or nonaromatic fluorescent or nonfluorescent group; Y is a hydrogen, alkyl, acetate, t-butyldimethylsilyl or an enzyme cleaveable group, or an antibody cleaveable group; R1 is selected from the group consisting of alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)2, alkyl(etheralkyl)3, alkyletherhaloalkyl, alkyl(etherhaloalkyl)2, alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenated alkyl(mono, di, tri or any position in normal or branched or cyclic chain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono or dibasic ) or the inorganic salts thereof, haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) or inorganic salts, linker-flourescent molecule, linker-antibodies, linker-antigen, linker-biotin, linker-avidin, linker-protein or linker-or linker-lipids; when R2 and R3 form 
xe2x80x83which is a cyclic or polycyclic alkyl group or spiro-fused ring with or without substitution or
(c). Ar is aryl and may be phenyl, substituted phenyl, naphthyl, substituted naphthyl, anthryl, substituted anthryl or any other aromatic or nonaromatic fluorescent or nonfluorescent group; Y is a hydrogen, alkyl, acetate, t-butyldimethylsilyl or an enzyme cleaveable group, or an antibody cleaveable group; R1 is selected from the group consisting of alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)2, alkyl(etheralkyl)3, alkyletherhaloalkyl, alkyl(etherhaloalkyl)2, alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenatedalkyl(mono, di, tri or any position in normal or branched or cyclic chain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono or dibasic ) or the inorganic salts thereof, haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) or inorganic salts, linker-flourescent molecule, linker-antibodies, linker-antigen, linker-biotin, linker-avidin, linker-protein or linker-carbohydrates or linker-lipids; where R2 and R3 are branched alkyl and cycloalkyl groups containing 3 to 8 carbon atoms which can contain halogens and hetero atoms in the ring or side chain thereof.
The new dioxetanes are triggered by the same activating agents described above.
The new alkenes hereof used to prepare the 1,2-dioxetanes hereof are prepared by the reaction of (a) 2-adamantanone or other spiro-fused ketone including ketones having a xcfx80-electron in the ring with (b) a substituted aromatic ester or ketone, using titanium trichloride or tetrachloride and a reducing agent such as an active metal or lithium aluminium hydride in tetrahydrofuran (THF) or other solvent of choice. This reaction is an intermolecular coupling of a ketone and an ester or ketone to form a vinyl ether using a modified McMurray procedure. Ordinarily, the reactants are present in at least stoichiometric quantities. However, excess amounts of the ester or ketone can be used. The temperatures at which the reactions as described above are those disclosed in the art.
Photooxygenation of the resulting vinyl ether by well-known conventional techniques affords 1,2-dioxetanes that are easily handled compounds with the desired stability.
The chemiluminescent decomposition of the 1,2-dioxetanes hereof, as noted above, can, preferably, can be conveniently triggered at room temperature by removing the protecting group with a fluoride ion, base or an enzyme to generate the unstable, aryloxide 1,2-dioxetane intermediate which cleaves to the starting materials and yields intense blue or other colored luminescence light.
For a more complete understanding of the present invention reference is made to the following detailed description and accompanying examples.
As stated above chemiluminescent enzyme substrates based on 1,2-dioxetane are well known in the literature for biological assays such as immunoassays and DNA probes. Use of these high energy compounds in biological systems requires 1,2-dioxetanes which are thermally stable at the temperature of the enzymatic reaction and which do not undergo rapid spontaneous decomposition in an aqueous buffer. The spiro-fused adamantyl dioxetanes hereof meet these requirements. The present 1,2-dioxetanes can be modified as substrates for various enzymes including aryl esterase, xcex2-galactosidase, alkaline phosphatase and others.
In accordance herewith and as noted above, the present invention provides new 1,2-dioxetanes.These new 1,2-dioxetanes hereof correspond to the formula: 
wherein
(1) when Arxe2x80x94Oxe2x80x94Y and OR join together to give an aryl group substituted with an X-oxy group to form a stable 1,2-dioxetane intermediate which is triggerable to form an unstable intermediate oxide, R2 and R3 either form 
xe2x80x83which is a cyclic, polycyclic or spiro-fused ring containing at least one carbon-carbon double bond or cabon-carbon triple bond in the ring or side chain with or without heteroatoms or 
xe2x80x83which is a cyclic, polycyclic or spiro-fused ring containing substituted or unsubstituted fused aromatic ring or substituted or unsubstituted aromatic ring attached by linker arms; or
(2) when Arxe2x80x94Oxe2x80x94Y and OR1 do not join together
(a) Ar is aryl and may be phenyl, substituted phenyl, naphthyl, substituted naphthyl, anthryl, substituted anthryl or other aromatic or nonaromatic fluorescent or nonfluorescent group; Y is a hydrogen, alkyl, acetate, t-butyldimethylsilyl or an enzyme cleaveable group, or an antibody cleaveable group; R1 is selected from the group consisting of alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)2, alkyl(etheralkyl)3, alkyletherhaloalkyl, alkyl(etherhaloalkyl)2, alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenated alkyl(mono, di, tri or any position in normal or branched or cyclic chain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono or dibasic ) or the inorganic salts thereof, haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) or inorganic salts, linker-flourescent molecule, linker-antibodies, linker-antigen, linker-biotin, linker-avidin, linker-protein or linker-carbohydrates or linker-lipids; when R2 and R3 form either 
xe2x80x83which is a cyclic, polycyclic or spiro-fused ring containing at least one carbon-carbon double bond or cabon-carbon triple bond in the ring or side chain with or without heteroatoms, or (ii) 
xe2x80x83which is a cyclic, polycyclic or spiro-fused ring containing substituted or unsubstituted fused aromatic ring or substituted or unsubstituted aromatic ring attached by linker arms, or
(b). Ar is aryl and may be phenyl, substituted phenyl, naphthyl, substituted naphthyl, anthryl, substituted anthryl or any other aromatic or nonaromatic fluorescent or nonfluorescent group; Y is a hydrogen, alkyl, acetate, t-butyldimethylsilyl or an enzyme cleaveable group, or an antibody cleaveable group; R1 is selected from the group consisting of alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)2, alkyl(etheralkyl)3, alkyletherhaloalkyl, alkyl(etherhaloalkyl)2, alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenated alkyl(mono, di, tri or any position in normal or branched or cyclic chain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono or dibasic) or the inorganic salts thereof, haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) or inorganic salts, linker-flourescent molecule, linker-antibodies, linker-antigen, linker-biotin, linker-avidin, linker-protein or linker-carbohydrates or linker-lipids; when R2 and R3 form 
xe2x80x83which is a cyclic, polycyclic alkyl group with or without substitution which are spiro-fused to the dioxetane ring, or
(c). Ar is aryl and may be phenyl, substituted phenyl, naphthyl, substituted naphthyl, anthryl, substituted anthryl or any other aromatic or nonaromatic fluorescent or nonfluorescent group; Y is a hydrogen, alkyl, acetate, t-butyldimethylsilyl or an enzyme cleaveable group, or an antibody cleaveable group; R1 is selected from the group consisting of alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)2, alkyl(etheralkyl)3, alkyletherhaloalkyl, alkyl(etherhaloalkyl)2, alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenatedalkyl(mono, di, tri or any position in normal or branched or cyclic chain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono or dibasic ) or the inorganic salts thereof, haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) or inorganic salts, linker-flourescent molecule, linker-antibodies, linker-antigen, linker-biotin, linker-avidin, linker-protein or linker-simple or complex carbohydrates or linker-simple and complex lipids; where R2 and R3 are branched alkyl and cycloalkylgroups containing 3 to 8 carbon atoms which can contain halogens and hetero atoms in the ring or side chain thereof.
Typically, the new alkenes hereof are prepared by the reaction of (a) 2-adamantanone or other spiro-fused ketone including ketones having a xcfx80-electron in the ring with (b) a substituted aromatic ester or ketone, using titanium trichloride or tetrachloride and a reducing agent such as an active metal or lithium aluminium hydride in tetrahydrofuran (THF). This reaction is an intermolecular coupling of a ketone and an ester or ketone to form a vinyl ether using a modified McMurray procedure. Ordinarily, the reactants are present in at least stoichiometric quantities. However, excess amounts of the ester or ketone can be used. The temperatures at which the reactions as described above are those disclosed in the art.
Photooxygenation of the resulting vinyl ether affords 1,2-dioxetanes that are easily handled compounds with the desired stability.
When these dioxetanes react with an activating reagent or agent which removes the Y moiety (formula 17), they decompose to form an aryl oxide 1,2-dioxetane intermediate of the formula: 
This aryl oxide oxides 1,2-dioxetane intermediate, then, spontaneously decomposes to produce light and compounds of the formulae: 
where compound (19) is the starting organic ketone and compound (20) is the residue of the starting organic ester or ketone when Ar Oxe2x88x92 and OR1 join together.
In practicing the present invention, compound (19) can be any one of or a mixture of adamantan-2-one, substituted adamantan-2-one, adamantan-2-one-4,5-ene, substituted adamantan-2-one-4,5-ene, 2-hydroxytricyclo[7.3.1.02,7]tridecan-13-one or substituted 2-hydroxytricyclo[7.3.1.02,7]tridecan-13-one, tricyclo[7.3.1.02,7]tridec-2,7-ene -13-one or substituted tricyclo[7.3.1.02,7]tridec-2,7-ene-13-one, bicyclo[3.3.1]nonan-9-one or substituted bicyclo[3.3.1]nonan-9-one benzonorbonen-7-one or substitutrd benzonorbornen-7-one, 2,4-dimethyl-3-propanone or substituted 2,4-dimethyl-3-propanone, dicyclopropyl ketone or substituted dicyclopropyl ketone, dicyclohexyl ketone or substituted dicyclohexyl ketone when compound (20) is selected from the group consisting of substituted or unsubstituted 9H-fluoren-9-one, 9H-xanthen-9-one, 2,2,2-trifluoroethyl 3-hydroxybenzoate or substituted 2,2,2-trifluoroethyl 3-hydroxybenzoate, 2-phenoxyethyl 3-hydroxybenzoate or substituted 2-phenoxyethyl 3-hydroxybenzoate, and the like further the ketone of formula (19) is selected from the group consisting of adamantan-2-one-4,5-ene, substituted adamantan-2-one-4,5-ene, tricyclo[7.3.1.02,7]tridec-2,7-ene-13-one, substituted tricyclo[7.3.1.02,7]tridec-2,7-ene-13-one when the compound of formula (20) is alkyl or aryl 3-hydroxybenzoate or substituted alkyl or aryl 3-hydroxybenzoate.
The alkenes hereof used to prepare the present dioxetane correspond to the formula: 
wherein R1, R2, R3 , Y and Ar are as described above,
These alkenes are prepared by the coupling of the above-described ketones and esters or ketones.
Generally, the intramolecular coupling reaction between the ketone and the ester or ketone is carried out at a temerature ranging from about 25xc2x0 C. to about 85xc2x0 C. and, perferably, from about 45xc2x0 C. to about 65xc2x0 C. A stocichiometric excess of either the ester or ketone may be used.
The coupling reaction is carried out in the presence of suitable solvents and active metals as described in the prior art denoted above, the disclosure above which are hereby incorporated by reference.
After the alkene is obtained it is then, photooxidized to form the stable, triggerable 1,2-dioxetane hereof. These dioxetanes can, then, be de-stablized or triggeredby the reaction with base, acid, enzyme and or inorganic or organic catalyst and or electron donor source in the presence or absence of a fluorscence compound, as described in the literature or above cited prior art.